Column chromatography is useful for both analytical and preparative separations, particularly for proteins and peptides. In analytical separations, the most efficient columns in many cases are high-performance liquid chromatography (HPLC), where a high back pressure (up to 6,000 psi) is applied to the column, and high resolution and reproducibility are sought. The binding capacity of the column is generally of little importance, and a high flow rate of sample through the column is generally used. Preparative separations are performed to extract purified proteins or peptides from a mixture rather than simply analyzing the mixture to determine its composition. While high resolution and reproducibility are still needed in preparative separations, the binding capacity and flow properties of the column are important as well, more so than in analytical columns. For preparative separations, therefore, it is important that high resolution be achieved with both a high binding capacity and a low back pressure (typically 1,000 psi or less).
High resolution requires both maximization of selectivity and minimization of band broadening. Factors that contribute to band broadening are longitudinal diffusion, eddy diffusion, and non-equilibrium mass transfer in both the mobile and stationary phases.
Longitudinal diffusion is of only minor concern for macromolecules. Eddy diffusion and non-equilibrium mass transfer in the mobile phase are of greater concern in packed beds, but both have been reduced by using columns packed with uniform particles with diameters in the 1- to 5-micron diameter range. Beds of this type, however, have high flow resistance. In addition, they are still susceptible to non-equilibrium mass transfer in the stationary phase, i.e., the diffusion of solute molecules into and out of the pores of the particles. This is a major contributing factor in band broadening, particularly with larger proteins due to their lower diffusion rates.
While diffusive mass transfer can be eliminated with the use of nonporous particles, the low surface area of such particles is detrimental to binding capacity. Surface area can be increased with the use of nonporous particles having diameters in the range of about 1 to 3 microns, but while the separations are rapid, particles of this size are not viable for preparative separations since they require a high back pressure. Another option is the use of perfusive particles, i.e., particles that contain both through-pores (6,000 to 8,000 .ANG.) that traverse the particles and are large enough to accommodate hydrodynamic flow, and diffusive pores of a much smaller diameter (500 to 1,500 .ANG.) that branch out from the through-pores. The through-pores however cause eddy diffusion, since the average linear velocity of mobile phase through a 7,000 .ANG. through-pore in a 10 micron diameter particle is only 5% of the average linear velocity through the interstices between the particles, and the difference in flow rate is the cause of eddy diffusion. Furthermore, while the intraparticle convective flow reduces the time required for intraparticle solute transport, the slow diffusive transport of solutes in the small-diameter branch pores still dominates the solute flow. This causes band broadening at high flow rates.
Particles were avoided entirely by the introduction of a macroporous solid plug spanning the cross section of the column (Netherlands Patent Application No. 6,803,739, Czechoslovakian Academy of Sciences, Prague, laid open to public inspection Sep. 17, 1969). The macroporous plug eliminated the need to prepare particles and pack them into columns; the plug was instead prepared by polymerization in the column itself. By using an organic solvent as a porogen, however, the resulting plug was macroporous, i.e., with pores having diameters of approximately 0.1 microns or less. Pores of this size cause the diffusive transport discussed above, which impedes hydrodynamic flow and results in band broadening. Furthermore, the use of an organic solvent required that polymerization be performed under anhydrous conditions, and that the plug be extensively washed prior to performing separations that used an aqueous mobile phase. A still further disadvantage is that the resulting bed was hydrophobic, which would reduce its usefulness in most cases.